Effect Of Skimmed-milk Powder And Soybean Lecithin Extender On Sheep Semen Preservation In Low Temperature

This study explored the feasibility of substituting skimmed-milk powder for milk and soy lecithin for yolk on sperm in low temperature, aiming to make proper dilution for sheep sperm in low temperature both in sheep research and in practice. Sheep sperm was collected using artificial vagina, fresh milk and yolk in sperm dilution were replaced by varying density of skimmed-milk powder and soybean lecithin respectively. Keep the dilution in low temperature, rewarm regularly and detected the sperm progressive motility, survival time, survival rate, abnormal rate and rate of acrosomal integrity to evaluate the effectiveness of sheep sperm in low temperature, and then prove it via in vitro fertilization(IVF).The test groups were added skimmed-milk powder(SMP)(4%, 7%, 10%, 13%) to replace fresh milk while the control group was added milk. The results showed that, for sperm progressive motility there was no difference among groups in the first 60 hours(P>0.05) except that the 10% SMP group was higher than other test groups. In 72~84h, the 10% SMP group was not different from the control group(P>0.05), but the 4%and 13% SMP groups were significantly lower than the control group(P<0.05). In 72~132h, the 4%SMP, 7%SMP and 13%SMP groups were lower than the 10% SMP group and the control group(P>0.05). For sperm survival rate, no differences were observed among groups in the first 72 hours(P>0.05). 10%SMP came to its peak at 96 h with a rate of 62.62%, but was not different from the control group(P>0.05). When it came to 120 h, 10%SMP was the highest among test groups(P<0.05), but with no remarkable differences compared with the control group(P<0.05). For abnormal sperm rate, neither 7% SMP nor 10% SMP was significantly different from the control group at 24 h, 72 h and 96h(P>0.05), and the 10% SMP group was the lowest. At 120 h, though similar with the control group(P>0.05), the 10% SMP group was remarkably different from other test groups(P<0.05). For the rate of acrosomal integrity, 10% SMP group was not different from the control group at 48h(P>0.05), but higher than the 7% SMP and 13% SMP group(P<0.05). Taking all the results into consideration, it’s best to replace fresh milk with 10% skimmed-milk powder.The test groups were added soybean lecithin(SBL)(0.375%, 0.750%, 1.500%, 3.000%) to replace yolk on the basis of 10% skimmed-milk powder optimized dilution. The results showed that, for sperm progressive motility, there was no difference between 0.375% SBL group and yolk group during 0~120h(P>0.05) and sperm motility maintained 0.43 in 0.375% SBL group at 84 h. During 60~96h, 1.500% SBL and 3% SBL group were lower than 0.375% SBL group and yolk group(P<0.05). While at 108 h, 0.750% SBL, 1.500% SBL and 3.000% SBL group were lower than yolk group(P<0.05). For sperm survival time, the 0.375% SBL group could survive 88 hours in effectiveness and 120 hours in total, which was not different from the yolk group(P>0.05). The rest of SBL groups were significantly lower than the 0.375% SBL group and yolk group(P<0.05). For sperm survival rate, no differences were observed among groups at 0h and 24h(P>0.05). From 48 h to 120 h, the 0.375% SBL group was similar with the yolk group(P>0.05). While at 72 h, 96 h and 120 h, the 0.375% SBL group was significantly higher than other SBL groups(P<0.05). For sperm abnormal rate, there was no differences between the 0.375% SBL group(P>0.05) and the yolk group and it went to the lowest for the 0.375% SBL group at 72 h and 96 h. For the rate of acrosomal integrity, there was no difference in SBL groups compared with the yolk group throughout the time(P>0.05). As can be concluded from all the evidences above, it’s best to replace yolk with 0.375% soybean lecithin.In vitro fertilization(IVF) of sheep was conducted using optimized sheep semen preservation in low temperature diluent(10% skimmed-milk powder to replace fresh milk and 0.375% soybean lecithin to replace yolk) with frozen sperm as comparison. The results showed that, cleavage rate in low temperature group was slightly higher than that in frozen group without significant differences(P>0.05, 63.12% and 55.92%, respectively), blastocysts development rate was remarkably higher in low temperature group than that in frozen group(P<0.05, 20.61% and 14.27%, respectively). There was no difference in the number of blastocyst cells after fluorescent staining between groups(P>0.05).